G proteins are key molecular switches that mediate cellular responses to a wide variety of signals including light, odor, taste, hormones, and neurotransmitters. Precise regulation of the intensity and duration of G protein-mediated signaling is critical for normal physiological responses as well as for disease prevention. The goal of this proposal is to uncover novel and fundamentally important mechanisms for G protein regulation, using the genetically tractable organism yeast as a model system. The main hypothesis is that G protein signaling is regulated via ubiquitination of G protein subunits. The hypothesis is based on recent observations in yeast that both G( subunit (Gpa1) and G( subunit (Ste4) are ubiquitinated, and mutants that block G protein ubiquitination profoundly affect signaling. Three specific aims are proposed: Aim 1: What is the functional role of G( Ste4 ubiquitination? A truncation mutant of Ste4 that is resistant to ubiquitination is hypersensitive to pheromone stimulation. Our hypothesis is that stimulus- dependent ubiquitination of Ste4 serves as a mechanism to down-regulate pheromone signaling. To test this, we will identify critical residues required for Ste4 ubiquitination and generate point-mutants that specifically block Ste4 ubiquitination. We will then determine the consequences of Ste4 ubiquitination by a detailed comparison of signaling, subcellular localization, binding ability and stability of wild type Ste4 versus point mutants of Ste4 that block its ubiquitination. Aim 2: How is G( Ste4 targeted for ubiquitination? Ste4 ubiquitination is induced upon pheromone stimulation. Our hypothesis is that pheromone-induced phosphorylation of Ste4 or its dissociation from the G( Gpa1 triggers its ubiquitination. To test this, we will examine Ste4 ubiquitination in Ste4 phosphorylation-sites mutants, mutants that lack kinase responsible for Ste4 phosphorylation, and mutants that lack the G( Gpa1. Alternatively, pheromone stimulation may directly activate the ubiquitinating enzymes. To test this, we will determine whether pheromone treatment alters activity of responsible E2 and E3 enzymes in vitro and in vivo. Aim 3: Why is Rsp5 required for the activation of G protein signaling? Disrupting E3 ligase Rsp5 in yeast severely diminishes signaling at the step of G protein. Gpa1 is mono-ubiquitinated by Rsp5 and has a positive signaling role at endosome. Our hypothesis is that Rsp5-catalyzed mono- ubiquitination is required for targeting Gpa1 to endosome to signal. This hypothesis will be tested by determining the effect of disrupting Rsp5 on endosomal localization and signaling of Gpa1. Alternative possibility that Rsp5-catalyzed ubiquitination may regulate G protein activation will also be tested. PUBLIC HEALTH RELEVANCE: Defects in G protein-mediated signaling pathways can lead to a variety of diseases and disorders including heart diseases, hypertension, blindness and endocrine disorders. This project will reveal novel mechanisms that regulate G protein signaling, which will provide significant insights for the understanding of disease mechanisms and aid in the development of new therapeutics.